Pteroyl-gamma-glutamate-cysteine synthesis and its application in folate receptor-mediated cancer cell targeting using folate-tethered liposomes

Cell membrane-associated folate receptors are selectively overexpressed in certain human tumors. The high affinity of folic acid for folate receptors provides a unique opportunity to use folic acid as a targeting ligand to deliver chemotherapeutic agents to cancer cells. Folate-tethered liposomes bearing pteroyl-gamma-glutamate-cysteine-polyethylene glycol (PEG)-distearoylphosphatidylethanolamine (DSPE) as the targeting component are under investigation as mediators of drug and gene delivery to cancer cells that overexpress folate receptors. Pteroyl-gamma-glutamate-cysteine synthesis is one of the crucial starting steps in the preparation of pteroyl-gamma-glutamate-cysteine-PEG-DSPE. However, published methods for the synthesis of pteroyl-gamma-glutamate-cysteine provide low yields and are not easily reproducible. Therefore, we developed a modified synthetic method for the removal of the N(10)-trifluoroacetyl group after cleavage/deprotection that is reliable, is easily reproducible, and has high yield (38%) compared with an unreliable yield of 3-20% with the earlier methods. Folate-tethered liposomes containing calcein or doxorubicin were prepared using pteroyl-gamma-glutamate-cysteine-PEG-DSPE as the targeting component along with nontargeted liposomes with PEG-DSPE. The results of the uptake of calcein and cytotoxicity of doxorubicin in human cervical cancer HeLa-IU(1) cells and human colon cancer Caco-2 cells demonstrated that folate-tethered liposomes were efficient in selective delivery to cancer cells overexpressing folate receptors. The improvement in yield of the targeting component can significantly facilitate "scale up" of folate receptor-mediated liposomal cancer therapy to the preclinical and clinical levels of investigations.     

Folic acid and pterin deaminases in Dictyostelium discoideum: kinetic properties and regulation by folic acid, pterin, and adenosine 3'',5''-phosphate.

Kinetic data obtained for deamination of pterin by the extracellular fraction from Dictyostelium discoideum yielded apparently linear Lineweaver-Burk plots for pterin. The Michaelis constant for pterin was 30 microM. The data for folic acid deamination yielded convex Lineweaver-Burk plots. Convex Lineweaver-Burk plots could result from the presence of two types of enzymes with different affinities. The data for folic acid deamination were analyzed mathematically for two types of enzymes. This analysis produced Michaelis constants for folic acid of 1.8 and 23 microM competition studies suggested that an enzyme with low affinity nonspecifically catalyzed the deamination of folic acid and pterin, whereas an enzyme with high affinity was a specific folic acid deaminase. A specific folic acid deaminase with high affinity appeared to be present on the surface of D. discoideum cells. The Michaelis constant for this enzyme was 2.6 microM. Cells growing in nutrient broth and cells starved in phosphate buffer released folic acid and pterin deaminases. The quantity of deaminase activities released by the cells appeared to be controlled by chemoattractants. Starving cells that were supplied with folic acid, pterin, or adenosine 3',5'-phosphate increased their extracellular folic acid and pterin deaminase activities to a larger extent than did cell suspensions to which no chemoattractants were added. Administration of folic acid or pterin to starving cells caused increases of the activity of extracellular adenosine 3',5'-phosphate phosphodiesterase and repressed increases of the activity of phosphodiesterase inhibitor.     

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.     

Design and synthesis of releasable folate-drug conjugates using a novel heterobifunctional disulfide-containing linker

Cellular uptake of vitamin folic acid occurs via folate-receptor mediated endocytosis. Many types of cancer cells express high levels of folate receptors as they need continuous supply of this vitamin for their proliferation. With an objective to use folic acid as a 'Trojan Horse' to transport anticancer drugs into cancer cells, a novel heterobifunctional disulfide-containing linker was synthesized and utilized to covalently link an amino- and hydroxyl-containing anticancer drug, and an appropriately functionalized folic acid to create novel targetable folate-drug conjugates that are shown to release free drugs under biologically relevant pH via sulfhydryl-assisted cleavage of the self-immolative disulfide-containing linker.     

Polyester dendritic systems for drug delivery applications: in vitro and in vivo evaluation

High molecular weight polymers (> 20 000 Da) have been widely used as soluble drug carriers to improve drug targeting and therapeutic efficacy. Dendritic polymers are exceptional candidates for the preparation of near monodisperse drug carriers due to their well-defined structure, multivalency, and flexibility for tailored functionalization. We evaluated various dendritic architectures composed of a polyester dendritic scaffold based on the monomer unit 2,2-bis(hydroxymethyl)propanoic acid for their suitability as drug carriers both in vitro and in vivo. These systems are both water soluble and nontoxic. In addition, the potent anticancer drug, doxorubicin, was covalently bound via a hydrazone linkage to a high molecular weight 3-arm poly(ethylene oxide)-dendrimer hybrid. Drug release was a function of pH, and the release rate was more rapid at pH < 6. The cytotoxicity of the DOX-polymer conjugate measured on multiple cancer lines in vitro was reduced but not eliminated, indicating that some active doxorubicin was released from the drug polymer conjugate under physiological conditions. Furthermore, biodistribution experiments show little accumulation of the DOX-polymer conjugate in vital organs, and the serum half-life of doxorubicin attached to an appropriate high molecular weight polymer has been significantly increased when compared to the free drug. Thus, this new macromolecular system exhibits promising characteristics for the development of new polymeric drug carriers.     

Cardiovascular abnormalities in Folr1 knockout mice and folate rescue

BACKGROUND: Periconceptional folic acid supplementation is widely believed to aid in the prevention of neural tube defects (NTDs), orofacial clefts, and congenital heart defects. Folate-binding proteins or receptors serve to bind folic acid and 5-methyltetrahydrofolate, representing one of the two major mechanisms of cellular folate uptake. METHODS: We herein describe abnormal cardiovascular development in mouse fetuses lacking a functional folate-binding protein gene (Folr1). We also performed a dose-response study with folinic acid and determined the impact of maternal folate supplementation on Folr1 nullizygous cardiac development. RESULTS: Partially rescued preterm Folr1(-/-) (formerly referred to as Folbp1) fetuses were found to have outflow tract defects, aortic arch artery abnormalities, and isolated dextracardia. Maternal supplementation with folinic acid rescued the embryonic lethality and the observed cardiovascular phenotypes in a dose-dependant manner. Maternal genotype exhibited significant impact on the rescue efficiency, suggesting an important role of in utero folate status in embryonic development. Abnormal heart looping was observed during early development of Folr1(-/-) embryos partially rescued by maternal folinic acid supplementation. Migration pattern of cardiac neural crest cells, genetic signals in pharyngeal arches, and the secondary heart field were also found to be affected in the mutant embryos. CONCLUSIONS: Our observations suggest that the beneficial effect of folic acid for congenital heart defects might be mediated via its impact on neural crest cells and by gene regulation of signaling pathways involved in the development of the pharyngeal arches and the secondary heart field.     

Radiosynthesis and evaluation of a 99mTc-folic acid radiotracer prepared using [99mTcN(PNP)]2+ metal fragment

Folate receptors (FR) are over-expressed on a wide variety of tumor cells and are a potential molecular target for radiolabeled folates. In this respect, several SPECT and PET based radiofolates have been evaluated in the past albeit with their high renal uptake posing limitation towards their clinical use. To overcome this, a new ^99mTc labeled folic acid was synthesized via the use of [^99mTcN(PNP)]²⁺ metal fragment, where the presence of the latter pharmacophore redirects in vivo clearance via the hepatobiliary pathway. In this respect, folic acid was derivatized at the γ-acid group with a cysteine BFCA (bifunctional chelating agent) and subsequently reacted with the preformed [^99mTcN]²⁺ intermediate in presence of PNP2 (bisphosphine) ligand, to yield the final complex. While preliminary, in vivo distribution of the complex exhibited high association of activity with liver and intestines and provided support to the rationality of the present design as clearance of labeled folic acid could be effected via the hepatic route, the in vitro studies of the folic acid-cysteine conjugate carried out in KB-31 cells, did not show much promise with reduction in receptor affinity in comparison with the native folic acid. The route followed herein to prepare a folic-acid based radiotracer constitutes the first report of radiolabeling folic acid using the [^99mTcN(PNP)]²⁺ as a radiosynthon. Modification in the structure of conjugate by linking the BFCA through a long-chain linker can be envisaged to improve the affinity of [^99mTcN(PNP)]-folic acid complex towards FRs.     

Folic acid conjugates of a bleomycin mimic for selective targeting of folate receptor positive cancer cells

A major challenge in the application of cytotoxic anti-cancer drugs is their general lack of selectivity, which often leads to systematic toxicity due to their inability to discriminate between malignant and healthy cells. A particularly promising target for selective targeting are the folate receptors (FR) that are often over-expressed on cancer cells. Here, we report on a conjugate of the pentadentate nitrogen ligand N4Py to folic acid, via a cleavable disulphide linker, which shows selective cytotoxicity against folate receptor expressing cancer cells.     

Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting

Here, we report the facile preparation of tunable magnetic Ni-doped near-infrared (NIR) quantum dots (MNIR-QDs) as an efficient probe for targeting, imaging, and cellular sorting applications. We synthesized the MNIR-QDs via a hot colloidal synthesis approach to yield monodisperse and tunable QDs. These hydrophobic QDs were structurally and compositionally characterized and further functionalized with amino-PEG and carboxyl-PEG to improve their biocompatibility. Since QDs are known to be toxic due to the presence of cadmium, we have evaluated the in vitro and in vivo toxicity of our surface-functionalized MNIR-QDs. Our results revealed that surface-functionalized MNIR-QDs did not exhibit significant toxicity at the concentrations used in the experiments and are therefore suitable for biological applications. For further in vitro applications, we covalently linked folic acid to the surface of amino-PEG-coated MNIR-QDs through NHS chemistry to target the folate receptors largely present in the HeLa cells to demonstrate the specific targeting and magnetic behavior of these MNIR-QDs. Improved specificity has been observed with treatment of HeLa cells with the folic acid-linked amino PEG-coated MNIR QDs (FA-PEG-MNIR-QDs) compared to the one without folic acid. Since the synthesized probe has magnetic property, we have also successfully demonstrated sorting between the cells which have taken up the probe with the use of a magnet. Our findings strongly suggest that these functionalized MNIR-QDs can be a potential probe for targeting, cellular sorting, and bioimaging applications.     

Is a metabolic control for the doubling of the macromolecule synthesizing machinery possible.

The usual models for cell multiplication only involve macromolecular events and ignore the influence of metabolism. The conjecture presented here shows how metabolism could be involved. Separate pathways might cooperate for the synthesis of a small molecule, acting as positive control of several macromolecular syntheses, including stable RNA synthesis. A detailed analysis of the available data showing an influence of metabolites on macromolecular syntheses suggests that two main pathways might cooperate, one involving serine and folic acid metabolism and the second involving threonine and the branched chain aminoacids. Experiments are suggested to challenge the conjecture.     

Effects of folate cycle disruption by the green tea polyphenol epigallocatechin-3-gallate

We demonstrate that the tea polyphenol, epigallocatechin-3-gallate, is an efficient inhibitor of human dihydrofolate reductase. Like other antifolate compounds, epigallocatechin-3-gallate acts by disturbing folic acid metabolism in cells, causing the inhibition of DNA and RNA synthesis and altering DNA methylation. Epigallocatechin-3-gallate was seen to inhibit the growth of a human colon carcinoma cell line in a concentration and time dependent manner. Rescue experiments using leucovorin and hypoxanthine–thymine medium were the first indication that epigallocatechin-3-gallate could disturb the folate metabolism within cells. Epigallocatechin-3-gallate increased the uptake of [³H]-thymidine and showed synergy with 5-fluorouracil, while its inhibitory action was strengthened after treatment with hypoxanthine, which indicates that epigallocatechin-3-gallate decreases the cellular production of nucleotides, thus, disturbing DNA and RNA synthesis. In addition to its effects on nucleotide biosynthesis, antifolate treatment has been linked to a decrease in cellular methylation. Here, we observed that epigallocatechin-3-gallate altered the p16 methylation pattern from methylated to unmethylated as a result of folic acid deprivation. Finally, we demonstrate that epigallocatechin-3-gallate causes adenosine to be released from the cells because it disrupts the purine metabolism. By binding to its specific receptors, adenosine can modulate different signalling pathways. This proposed mechanism should help us to understand most of the molecular and cellular effects described for this tea polyphenol.     

Identification of folate binding macromolecule in rabbit choroid plexus.

A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.     

饲料中添加叶酸和VB12对中华绒螯蟹幼蟹生长、非特异性免疫和抗病力的影响

为研究叶酸和VB12协同作用对中华绒螯蟹(Eriocheir sinensis)幼蟹生长、非特异性免疫和抗病力的影响,选取初始体重为(2.570.03) g的幼蟹600只,随机分成4组,每组5个重复,每个重复30只幼蟹,分别投喂对照组(不添加叶酸和VB12),单一VB12组(0.2 mg/kg),单一叶酸组(2.3 mg/kg)和联合处理组(0.2 mg/kg VB12 +2.3 mg/kg叶酸)的饲料8周。在养殖实验结束后,先统计成活率和称重,然后从每个处理组随机选取30只幼蟹,用2108 CFU/mL的嗜水气单胞菌注射攻毒2周。实验结果表明:幼蟹的增重率、特定生长率、饲料效率和存活率在联合处理组最高,显著高于对照组(P0.05),但与单一叶酸或VB12组相比不存在显著差异(P0.05)。联合处理组的血清酚氧化酶活性显著高于对照组(P0.05),但与单一叶酸或VB12组也无显著性差异(P0.05)。同时,联合处理组的血清酸性磷酸酶、碱性磷酸酶、溶菌酶活性和血细胞总数等指标最高,其次是单一叶酸组和VB12组,而对照组最低。投喂联合处理组饲料幼蟹的肝胰腺超氧化物歧化酶活性最高,而丙二醛含量和累积死亡率最低。以上结果表明,叶酸和VB12对幼蟹的生长、生理代谢和免疫性能均可能有互补和协同作用,养殖生产中建议饲料中叶酸和VB12添加量分别为2.3 mg/kg和0.2 mg/kg。       

Folic Acid Conjugated δ-Valerolactone-Poly(ethylene glycol) Based Triblock Copolymer as a Promising Carrier for Targeted Doxorubicin Delivery

The aim of this study is to test the hypothesis that the newly synthesized poly(δ-valerolactone)/poly(ethylene glycol)/poly(δ-valerolactone) (VEV) copolymer grafted with folic acid would impart targetability and further enhance the anti-tumor efficacy of doxorubicin (DOX). Here, folic acid conjugated VEV (VEV-FOL) was synthesized by a modified esterification method and characterized using IR and NMR. DOX loaded VEV-FOL micelles were synthesized using a novel solvent evaporation method and were obtained with a mean diameter of 97 nm with high encapsulation efficiency and sustained in vitro release profile. Comparative studies of polymer micelles with and without folate for cellular uptake and cytotoxicity were done on folate receptor-positive breast cancer cell line, MDAMB231. The intracellular uptake tests showed significant increase in folate micellar uptake when compared to non-folate-mediated micelles. MTT assay followed by apoptosis assays clearly indicated that folate decorated micelles showed significantly better cytotoxicity (IC₅₀ = 0.014 µM) and efficiency to induce apoptosis than other treated groups. Moreover, a significant G2/M arrest was induced by DOX loaded VEV-FOL micelles at a concentration where free drug failed to show any activity. Thus, our results show that the folic acid-labeled VEV copolymer is a promising biomaterial with controlled and sustainable tumor targeting ability for anticancer drugs which can open new frontiers in the area of targeted chemotherapy.     

Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction

Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.     

Design, synthesis, and biological evaluation of folic acid targeted tetraphenylporphyrin as novel photosensitizers for selective photodynamic therapy

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate light wavelength, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Targeted PDT offers the opportunity of enhancing photodynamic efficiency by directly targeting diseased cells and tissues. Two new conjugates of three components, folic acid/hexane-1,6-diamine/4-carboxyphenylporphyrine 1 and folic acid/2,2'-(ethylenedioxy)-bis-ethylamine/4-carboxyphenylporphyrine 2 were synthesized. The conjugates were characterized by 1H NMR, MALDI, UV-visible spectroscopy, and fluorescence quantum yield. The targeted delivery of these photoactive compounds to KB nasopharyngeal cell line, which is one of the numerous tumor cell types that overexpress folate receptors was studied. It was found that after 24 h incubation, conjugates 1 and 2 cellular uptake was on average 7-fold higher than tetraphenylporphyrin (TPP) used as reference and that 1 and 2 cellular uptake kinetics increased steadily over the 24 h period, suggesting an active transport via receptor-mediated endocytosis. In corresponding results, conjugates 1 and 2 accumulation displayed a reduction of 70% in the presence of a competitive concentration of folic acid. Survival measurements demonstrated that KB cells were significantly more sensitive to conjugated porphyrins-mediated PDT. Under the same experimental conditions and the same photosensitizer concentration, TPP displayed no photocytotoxicity while conjugates 1 and 2 showed photodynamic activity with light dose values yielding 50% growth inhibition of 22.6 and 6.7 J/cm2, respectively.     

GSH-responsive anti-mitotic cell penetrating peptide-linked podophyllotoxin conjugate for improving water solubility and targeted synergistic drug delivery

Podophyllotoxin (PPT) is a chemotherapeutic agent which has shown significant anti-cancer effects through inhibiting microtubule assembly. However, because of the poor water solubility and obvious side effects, PPT cannot be used in clinical cancer therapy. In order to solve these problems, a novel glutathione-responsive PPT conjugate has been synthesized in which PPT was linked to an anti-mitotic cell penetrating peptide (PRA) via a disulfide linkage. In particular, the as-prepared PPT-PRA conjugate can self-assemble into vesicle in water, furthermore, another anti-cancer drug (doxorubicin was chosen as an example) can be loaded in the vesicle for synergistic drug delivery. For better cancer cells targeting, the vesicle was then modified with folic acid (FA). The results indicated that the as-prepared FA modified drug-loaded vesicle not only could overcome the poor water solubility and side effects of PPT but also exhibited targeted toxicity and synergistic therapeutic effect.     

An intervention study to increase knowledge and use of folic acid among relatives in neural tube defect-affected families in Washington, D.C

BACKGROUND: Little is known about the level of knowledge and use of folic acid among near relatives in U.S. families of a child with spina bifida. We hypothesized that relatives would be more knowledgeable than the general population and more likely to take folic acid. Further, we hypothesized that relatives would be more motivated by an intervention to increase their use of folic acid. METHODS: We conducted an intervention study among females in families attending a hospital spina bifida clinic in Washington, DC. RESULTS: The 231 subjects consisted of the affected individuals, mothers, sisters, and aunts. The average age was 34 years. At baseline, most (87.4%) reported that they had heard of folic acid; 37.6% were currently taking multivitamins with folic acid and 6.9% were taking folic acid tablets. The intervention significantly increased both knowledge (to 99%) and intake of folic acid from 41.9 to 48.5%. Folic acid intake increased significantly among African-American women and women with less education, women who were older, married, with children, and nonsmokers. CONCLUSIONS: This intervention was successful in increasing folic acid intake among female relatives in spina bifida-affected families. By the end of the study, almost all women had heard of folic acid and folic acid use had increased by 16%. Among these women at higher than expected risk for having an affected child, this rate of intake, while more than the general population, still falls short of optimum. Fortification of food with folic acid may be the only way to ensure increased folic acid intake.     

Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A

Macrophage scavenger class A type I and type II receptors (SR-A) are trimeric, integral membrane glycoproteins that bind an unusually broad array of macromolecular ligands. These ligands include modified proteins and lipoproteins, nucleic acids, and a variety of plant and microbial cell wall constituents, such as fucoidan and lipoteichoic acid. Early studies of SR-A functions indicated that the receptors bound, internalized, and degraded their ligands without provoking any macrophage activating signaling events. More recent studies have provided evidence that several SR-A ligands can activate macrophage gene expression via utilization of a receptor-linked, PI3-kinase pathway. To investigate the role of SR-A in engaging signal transduction events, we employed macrophages taken from mice lacking these receptors. Using either fucoidan or lipoteichoic acid, we confirm that both ligands stimulate tyrosine phosphorylation of PI3-kinase and production of modest levels of the cytokine, TNFalpha. However, macrophages taken from SR-A null mice did not differ from wild type macrophages in these responses, indicating that these signaling events arise independently of SR-A activity. Employing mice lacking CD14, a GPI anchored receptor that binds bacterial lipopolysaccharide and signals via activation of Toll-like receptors, we show that the fucoidan and lipoteichoic acid responses are largely abrogated when CD14 is absent. These data do not provide support for direct SR-A involvement in signal transduction events and suggest that the early characterization of these receptors as initiators of a non-phlogistic, pathogen clearance pathway was correct.     

Studying the mechanism of sperm DNA damage caused by folate deficiency

Sperm DNA injury is one of the common causes of male infertility. Folic acid deficiency would increase the methylation level of the important genes, including those involved in DNA double‐strand break (DSB) repair pathway. In the early stages, we analysed the correlation between seminal plasma folic acid concentration and semen parameters in 157 infertility patients and 91 sperm donor volunteers, and found that there was a significant negative correlation between seminal folic acid concentration and sperm DNA Fragmentation Index (DFI; r = −0.495, p < 0.01). Then through reduced representation bisulphite sequencing, global DNA methylation of sperm of patients in the low folic acid group and the high folic acid group was analysed, it was found that the methylation level in Rad54 promoter region increased in the folic acid deficiency group compared with the normal folic acid group. Meanwhile, the results of animal model and spermatocyte line (GC‐2) also found that folic acid deficiency can increase the methylation level in Rad54 promoter region, increased sperm DFI in mice, increased the expression of γ‐H2AX, that is, DNA injury marker protein, and increased sensitivity of GC‐2 to external damage and stimulation. The study indicates that the expression of Rad54 is downregulated by folic acid deficiency via DNA methylation. This may be one of the mechanisms of sperm DNA damage caused by folate deficiency.